The ability for bacteria to produce chronic infections has been linked to the ability to produce biofilms. Biofilms are intrinsically resistant to both antibiotics and host-derived antimicrobial strategies, which can make treatment nearly impossible. Biofilm populations are also genetically and phenotypically diverse, which can undermine uniform antimicrobial strategies. Yet the role of this diversity in biofilm-related disease remains unclear. One major question is whether biofilm diversity arises from i) transient physiological variability among genetically identical individuals or ii) from the evolution of stable, genetically distinct subpopulations adapted to different biofilm microniches. To test these non-exclusive alternative hypotheses, we experimentally evolved replicate populations of the opportunistic, biofilm-forming pathogen Burkholderia cenocepacia under conditions favoring biofilm formation or planktonic growth. Our preliminary results support both models. First, the ancestor and evolved mutants each acclimate over time in various ways to form better biofilms when surface adhesion is favored, demonstrating physiological plasticity. Second, we observe repeated, heritable evolutionary diversification within biofilm-evolved populations but not within planktonic-evolved populations. These mutants are functionally distinct, produce superior biofilms than the ancestor, mimic the colony types frequently recovered from chronic infections, and interact synergistically in biofilms. To address the relative importance of these alternative models, we will first compare how acclimation and evolutionary adaptation affect the antimicrobial resistance and pathogenic potential of B. cenocepacia biofilms. Pathogenesis will be modeled using modified C. elegans virulence assays and by quantifying growth and cytotoxicity on CFTR lung epithelial cell lines. We are specifically interested in how different mutants within the community affect resistance and virulence, and whether certain types or combinations retain the potential for acute infection. We will also identify the mutational mechanisms that produce biofilm diversity and synergy by llumina genome resequencing, which will yield a small subset of functionally relevant mutations within a larger pool of false- positives. We will use conventional sequencing to eliminate false positives and then evaluate effects of the bona fide mutations by complementation and knockout studies in ancestral and mutant strains. Consistent with the AREA mechanism, each of these projects offers a range of training opportunities for graduate and undergraduate students. In summary, our long-range goal is to identify the molecular and physiological mechanisms critical for adaptation to the biofilm lifestyle, which would provide powerful targets for therapeutics. PUBLIC HEALTH RELEVANCE: Bacteria living on surfaces alter their lifestyle by clustering and covering themselves with slime that both protects them and affixes them to the surface. These communities are known as biofilms and are typified by high resistance to antibiotics and exceptional diversity among cells. This project seeks to identify the root causes of this lifestyle switch that can produce persistent, untreatable infections.